Biomedical Engineering Reference
In-Depth Information
The strategy for smaller proteins is to try to assign as many of the observable
resonances as possible to unique nuclei in the protein. As protein size increases
this objective becomes increasingly harder to obtain. The traditional suite of
multi-dimensional heteronuclear NMR experiments that facilitate backbone
resonance assignment cease to be effective for proteins above 50-100 kDa,
even when perdeuteration is employed. As discussed in Section 1.1.2, the
largest single-chain polypeptide for which near-complete backbone resonance
assignments have been made is MSG (82 kDa). For this protein, methyl
resonances were assigned using previously determined backbone resonance
assignments and NMR experiments that correlate backbone and methyl group
nuclei.
36
Obtaining resonance assignment for methyl groups of much larger
proteins where backbone resonance assignments are unavailable requires
alternative strategies. Several examples of different methyl group assignment
procedures that have been successfully applied to very large proteins (.100
kDa) are provided below. These approaches have lead to near-complete
assignment of detectable methyl resonances in the full-size protein in its native
oligomeric state.
1.3.1
Resonance Assignment of Large Proteins by 'Divide and
Conquer'
1.3.1.1 Deconstruction/Reconstruction of Large Oligomers
To date, many of the supramolecular protein systems studied by NMR
spectroscopy have been multimeric (Figure 1.1). A number of features of large
homo- or hetero-oligomeric proteins make them attractive for NMR studies.
Large homo-oligomers composed of smaller subunits have a high degree of
internal symmetry that serves to greatly reduce the complexity of NMR spectra
as a given nucleus will be in the same chemical environment in each subunit
and will thus have the same chemical shift. Furthermore, the size of the
monomeric subunit is often sufficient to allow the use of traditional backbone-
based resonance assignment strategies. However, within the context of the
quaternary complex the retarded tumbling rate means that canonical NMR
assignment experiments do not work.
One solution to this problem has been to try to dismantle the quaternary
complex into smaller, more NMR-friendly sized pieces (Figure 1.6). With the
aid of high-resolution 3D structures it is possible to predict and design
mutations that disrupt the formation of key oligomeric interfaces. If suitable
mutations can be found, and providing the smaller species remains folded and
stable, more standard NMR approaches can be applied.
The 'divide-and-conquer' technique was applied to the 20S oligomeric
proteasome particle in a landmark study by Sprangers and Kay.
45
The 20S
proteasome is composed of two subunits, a and b, both of which form seven-
mer rings that arrange into a 670 kDa a
7
b
7
b
7
a
7
quaternary structure
(Figure 1.1). Despite the considerable size of this complex, excellent quality
